PROJECT SUMMARY Mitochondria are essential organelles responsible for many cellular processes including energy production through oxidative phosphorylation (OXPHOS), calcium signaling, redox homeostasis, and cell death. Many of these functions are executed by a densely packed network of proteins in the inner mitochondrial membrane (IMM). As such, mitochondrial functions depend on protein homeostasis (proteostasis) in the IMM. IMM proteostasis is maintained by an extensive protein quality control system composed, in part, of IMM proteases that degrade misfolded proteins. Persistent IMM protein misfolding is speculated to cause a growing number of age-dependent neurodegenerative and neuromuscular degenerative diseases. For example, mutations in IMM proteases cause Parkinson's disease, spinocerebellar ataxia, spastic ataxia syndrome, spastic paraplegia, and other neurodegenerative diseases. Does a failure to degrade misfolded IMM proteins cause these diseases? This is unknown. In addition, misfolded variants of the ADP/ATP exchanger in the IMM, Ant1, cause autosomal dominant Progressive External Ophthalmoplegia and a mitochondrial myopathy. We used one such misfolded Ant1 variant to dissect the cellular consequences of IMM protein misfolding in yeast. Unexpectedly, IMM protein misfolding does not kill yeast cells by affecting essential mitochondrial functions in the IMM, but instead by disrupting mitochondrial protein import causing the toxic accumulation of mitochondrial precursor proteins in the cytosol. This novel mechanism was termed mitochondrial precursor overaccumulation stress (mPOS). It is not known if mPOS occurs in higher organisms, or if it can contribute to disease. Moreover, despite the significant clinical implications, the physiological consequences of IMM protein misfolding are poorly understood. To address these issues, we generated a novel knock-in (KI) mouse model expressing a pathogenic misfolded variant of Ant1. A fraction of KI mice undergo drastic neurodegeneration culminating in paralysis, thus confirming the pathogenic potential of IMM protein misfolding and providing a tool for in vivo mechanistic studies. Non-paralyzed KI mice exhibit exercise intolerance and reduced skeletal muscle mitochondrial respiration in skeletal muscle. This raises the possibility that IMM protein misfolding causes damage to specific components in the OXPHOS pathway leading to muscle weakness. We test this hypothesis in Aim 1. In addition to OXPHOS deficiency, cytosolic protein degradation pathways are activated in skeletal muscle of ?asymptomatic? KI mice. The activation of cytosolic protein degradation pathways suggests that cytosolic proteostasis is challenged, possibly via mPOS. Therefore, in Aim 2, we test the hypothesis that IMM protein misfolding induces mPOS in KI mouse muscle to cause muscle weakness. The mechanism(s) revealed in this proposal will be important for understanding the role of IMM protein misfolding in Ant1-induced pathologies and neuromuscular degenerative diseases alike. In long term, interventions alleviating the identified mechanism(s) may be effective treatments for these diseases, of which there are currently none. !